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An essential role of the autophagy activating kinase ULK1 in snRNP biogenesis.

ABSTRACT: The biogenesis of small uridine-rich nuclear ribonucleoproteins (UsnRNPs) depends on the methylation of Sm proteins catalyzed by the methylosome and the subsequent action of the SMN complex, which assembles the heptameric Sm protein ring onto small nuclear RNAs (snRNAs). In this sophisticated process, the methylosome subunit pICln (chloride conductance regulatory protein) is attributed to an exceptional key position as an 'assembly chaperone' by building up a stable precursor Sm protein ring structure. Here, we show that-apart from its autophagic role-the Ser/Thr kinase ULK1 (Uncoordinated [unc-51] Like Kinase 1) functions as a novel key regulator in UsnRNP biogenesis by phosphorylation of the C-terminus of pICln. As a consequence, phosphorylated pICln is no longer capable to hold up the precursor Sm ring structure. Consequently, inhibition of ULK1 results in a reduction of efficient UsnRNP core assembly. Thus ULK1, depending on its complex formation, exerts different functions in autophagy or snRNP biosynthesis.

SUBMITTER: Schmitz K

PROVIDER: S-EPMC8216288 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Saturated fatty acid (SFA)-induced lipotoxicity is caused by the accumulation of reactive oxygen species (ROS), which is associated with damaged mitochondria. Moreover, lipotoxicity is crucial for the progression of nonalcoholic steatohepatitis (NASH). Autophagy is required for the clearance of protein aggregates or damaged mitochondria to maintain cellular metabolic homeostasis. The NFE2L2/NRF2 (nuclear factor, erythroid 2 like 2)-KEAP1 (kelch like ECH associated protein 1) pathway is essential for the elimination of ROS. ULK1 (unc-51 like autophagy activating kinase 1; yeast Atg1) is involved in the initiation of autophagy; however, its role in lipotoxicity-induced cell death in hepatocytes and mouse liver has not been elucidated. We now show that ULK1 potentiates the interaction between KEAP1 and the autophagy adaptor protein SQSTM1/p62, thereby mediating NFE2L2 activation in a manner requiring SQSTM1-dependent autophagic KEAP1 degradation. Furthermore, ULK1 is required for the autophagic removal of damaged mitochondria and to enhance binding between SQSTM1 and PINK1 (PTEN induced kinase 1). This study demonstrates the molecular mechanisms underlying the cytoprotective role of ULK1 against lipotoxicity. Thus, ULK1 could represent a potential therapeutic target for the treatment of NASH.Abbreviations: ACTB: actin beta; CM-H2DCFDA:5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate; CQ: chloroquine; CUL3: cullin 3; DMSO: dimethyl sulfoxide; GSTA1: glutathione S-transferase A1; HA: hemagglutinin; Hepa1c1c7: mouse hepatoma cells; HMOX1/HO-1: heme oxygenase 1; KEAP1: kelch like ECH associated protein 1; LPS: lipopolysaccharides; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAPK8/JNK: mitogen-activated protein kinase 8; MEF: mouse embryonic fibroblast; MFN1: mitofusin 1; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NASH: nonalcoholic steatohepatitis; NFE2L2/NRF2: nuclear factor, erythroid 2 like 2; NQO1: NAD(P)H quinone dehydrogenase 1; PA: palmitic acid; PARP: poly (ADP-ribose) polymerase 1; PINK1: PTEN induced kinase 1; PRKAA1/2: protein kinase AMP-activated catalytic subunits alpha1/2; PRKN/PARK2: parkin RBR E3 ubiquitin protein ligase; PRKC/PKC: protein kinase C; RBX1: ring-box 1; ROS: reactive oxygen species; SFA: saturated fatty acid; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; TOMM20: translocase of outer mitochondrial membrane 20; TUBA: tubulin alpha; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; ULK1: unc-51 like autophagy activating kinase 1.

Project description:Although macroautophagy/autophagy is involved in hepatocellular carcinoma (HCC) initiation and development and has been identified as a mechanism of HCC therapy resistance, the role of ULK1 (unc-51 like autophagy activating kinase 1) in HCC remains unclear. Here, we report that both knockdown and knockout of ULK1 inhibited human HCC cell proliferation and invasion, and Ulk1 deletion abrogated tumor growth in a xenograft mouse model. Furthermore, ULK1 ablation in combination with sorafenib significantly inhibited HCC progression compared with sorafenib treatment alone or vehicle control. To identify candidate ULK1 inhibitors, we used a structure-based virtual docking approach to screen 3428 compounds. Among these compounds, XST-14 showed the highest affinity for the ULK1 protein and specifically blocked ULK1 kinase activity. Moreover, the Lys46, Tyr94 and Asp165 amino acid residues of ULK1 were required for its binding to XST-14 according to molecular docking and mutagenesis experiments. Functional assays revealed that XST-14 blocked autophagy and subsequently induced apoptosis and inhibited growth in HCC cells. More importantly, XST-14 acted synergistically with sorafenib to attenuate HCC progression by inhibiting sorafenib-induced autophagy activation both in vitro and in vivo. In addition, XST-14 was well tolerated and exhibited favorable drug metabolism and pharmacokinetic properties and low toxicity in mice. In summary, our study determined that ULK1 may represent a new therapeutic target for HCC and that targeting ULK1 in combination with sorafenib treatment may serve as a promising interventional strategy for treating HCC. Abbreviations: 3MA: 3-methyladenine; ADV: AutoDock Vina; ATP: adenosine triphosphate; EdU: 5-ethynyl-2'-deoxyuridine; ESI: electrospray ionization; HCC: hepatocellular carcinoma; IC50: half maximal inhibitory concentration; KD: kinase domain; q.o.d., every other day; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SPR, surface plasmon resonance.

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An essential role of the autophagy activating kinase ULK1 in snRNP biogenesis.

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